DNA replication termination in Escherichia coli parB (a dnaG allele), parA, and gyrB mutants affected in DNA distribution.

We investigated the Escherichia coli mutants carrying the parB, parA, and gyrB mutations, all of which display faulty chromosome partitioning at the nonpermissive temperature, to see whether their phenotype reflected a defect in the termination of DNA replication. In the parB strain DNA synthesis slowed down at 42 degrees C and the SOS response was induced, whereas in the parA strain DNA synthesis continued normally for 120 min and there was no SOS induction. To see whether replication forks accumulated in the vicinity of terC at the nonpermissive temperature, the mutants were incubated for 60 min at 42 degrees C and then returned to low temperature and pulse-labeled with [3H]thymidine. In all cases the restriction pattern of the labeled DNA was incompatible with that of the terC region, suggesting that replication termination was normal. In the parA mutant no DNA sequences were preferentially labeled, whereas in the parB and gyrB strains there was specific labeling of sequences whose restriction pattern resembled that of oriC. In the case of parB this was confirmed by DNA-DNA hybridization with appropriate probes. This test further revealed that the parB mutant over initiates at oriC after the return to the permissive temperature. Like dna(Ts) strains, the parB mutant formed filaments at 42 degrees C in the absence of SOS-associated division inhibition, accompanied by the appearance of anucleate cells of nearly normal size (28% of the population after 3 h), as revealed by autoradiography. The DNA in the filaments was either centrally located or distributed throughout. The parB mutation lies at 67 min, and the ParB- phenotype is corrected by a cloned dnaG gene or by a plasmid primase, strongly suggesting that parB is an allele of dnaG, the structural gene of the E. coli primase. It is thus likely that the parB mutant possesses an altered primase which does not affect replication termination but causes a partial defect in replication initiation and elongation and in chromosome distribution.     

Strong cellular preference in the expression of a housekeeping gene of Arabidopsis thaliana encoding S-adenosylmethionine synthetase.

S-Adenosylmethionine serves as a methyl group donor in numerous transmethylation reactions and plays a role in the biosynthesis of polyamines and ethylene. We have cloned and sequenced an S-adenosylmethionine synthetase gene (sam-1) of Arabidopsis thaliana. The deduced polypeptide sequence of the enzyme has extensive homology with the corresponding enzymes of Escherichia coli and yeast. Genomic hybridization indicates the presence of two adenosylmethionine synthetase genes per haploid Arabidopsis genome. RNA gel blot analysis shows that adenosylmethionine synthetase mRNA levels are high in stems and roots, correlating well with the higher enzyme activity in stems, compared with leaves. Histochemical analysis of transgenic Arabidopsis plants transformed with a chimeric beta-glucuronidase gene, under the control of 748-base pair 5' sequences of the sam-1 gene, demonstrates that the gene is expressed primarily in vascular tissues. In addition, high expression was observed in sclerenchyma and in the root cortex. A hypothesis for the strong cellular preference in the expression of the sam-1 gene is presented.     

Plant expression vectors with the origin of replication of the W-type plasmid Sa

A new class of binary vectors has been constructed, containing the origin of replication of the W-type plasmid Sa. These vectors are designed to express foreign genes in plants under control of the TR gene 2' promoter or the promoter of a light-inducible ribulose-1,5-bisphosphate carboxylase small subunit gene of Arabidopsis thaliana.     

Primary structure of a hormonally regulated beta-glucanase of Nicotiana plumbaginifolia

A cDNA clone for a hormonally regulated beta-glucanase from Nicotiana plumbaginifolia has been isolated by using an oligodeoxynucleotide probe, synthesized to match the previously determined N-terminal amino acid sequence. The cDNA has the complete sequence of the mature protein and contains at least part of a hydrophobic signal peptide. At the amino acid level, the beta-glucanase of N. plumbaginifolia is 73% homologous to a beta(1,3)-glucanase from tobacco and 52% homologous to a beta(1,3;1,4)-glucanase from barley. Southern-blot analysis clearly demonstrated that N. plumbaginifolia contains at least two related genes encoding beta-glucanase. The extent of the complete signal peptide of the cloned beta-glucanase was determined by sequencing part of the corresponding gene. Northern analysis showed that the expression of the beta-glucanase gene is influenced by auxins and cytokinins.     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

A binary vector for transferring genomic libraries to plants.

The transformation of mutant plants with a complete recombinant library derived from wild-type DNA followed by assay of transformed plants for complementation of the mutant phenotype is a promising method for the isolation of plant genes. The small genome of Arabidopsis thaliana is a good candidate for attempting this so-called shotgun transformation. We present the properties of an A. thaliana genomic library cloned in a binary vector, pC22. This vector, designed to introduce genomic libraries into plants, contains the oriV of the Ri plasmid pRiHR1 by which it replicates perfectly stably in Agrobacterium. Upon transfer of the library from E. coli to A. tumefaciens large differences in transfer efficiencies of individual recombinant clones were observed. There is a direct relation between transfer efficiency and stability of the recombinant clones both in E. coli and A. tumefaciens. The stability is independent of the insert size, but seems to be related to the nature of the insert DNA. The feasibility of shotgun transformation and problems of statistical sampling are discussed.     

The induction of manganese superoxide dismutase in response to stress in Nicotiana plumbaginifolia.

Superoxide dismutases (SODs) are metalloproteins that catalyse the dismutation of superoxide radicals to oxygen and hydrogen peroxide. The enzyme has been found in all aerobic organisms examined, where it plays a major role in the defence against toxic reduced oxygen species which are generated in many biological oxidations. Here we report the complete primary structure of a plant manganese superoxide dismutase (MnSOD), deduced from a cDNA clone of Nicotiana plumbaginifolia. The plant protein is highly homologous to MnSODs from other organisms and also contains an N-terminal leader sequence resembling a transit peptide for mitochondrial targeting. The location of the mature protein within the mitochondria has been demonstrated by subcellular fractionation experiments. We have analysed the expression profile of this MnSOD and found that it is dramatically induced during stress conditions, most notably in tissue culture as a result of sugar metabolism and also as part of the pathogenesis response of the plant, being induced by ethylene, salicylic acid, and Pseudomonas syringae infection. This induction is always accompanied by an increase in cytochrome oxidase activity, which suggests a specific protective role for MnSOD during conditions of increased mitochondrial respiration.